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Excess polycation mediates efficient chitosan-based gene transfer by promoting lysosomal release of the polyplexes.

Abstract：

:The optimal ratio of the polycation's amine to DNA phosphate group (N:P) for efficient polymer-based transfection always employs excess polycation versus DNA. Most of the excess polycation remains free in solution, unassociated with the polyplexes, but is essential for efficient transfection. The mechanism by which excess polycation increases transfection efficiency is not identified. We hypothesised that excess chitosan facilitates intracellular lysosomal escape of the polyplexes. We highlight here the essential role of excess chitosan by rescuing poorly transfecting low N:P ratio polyplexes, by adding free chitosan before or after polyplex addition to cells. We examined polyplex uptake, the kinetics of rescue, intracellular trafficking, and the effects of lysosomotropic agents. We found the facilitating role of excess chitosan to be downstream of cellular uptake. Live-cell confocal quantification of intracellular trafficking revealed prolonged colocalisation of low N:P polyplexes within lysosomes, compared to shorter residence times for both rescued or N:P 5 samples, followed by observation of free pDNA in the cytosol. These data demonstrate that excess polycation mediates enhanced transfection efficiency by promoting the release of polyplexes from the endo-lysosomal vesicles, revealing a critical intracellular barrier overcome by excess polycation and suggesting possible avenues for further optimisation of polymer-based gene delivery.

journal_name

Biomaterials

journal_title

Biomaterials

authors

Thibault M,Astolfi M,Tran-Khanh N,Lavertu M,Darras V,Merzouki A,Buschmann MD

doi

10.1016/j.biomaterials.2011.03.010

subject

Has Abstract

pub_date

2011-07-01 00:00:00

pages

4639-46

issue

20

eissn

0142-9612

issn

1878-5905

pii

S0142-9612(11)00250-X

journal_volume

32

pub_type

杂志文章

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